PROJECT SUMMARY/ABSTRACT Tuberculosis (TB) is the leading infectious cause of death worldwide. There is an urgent need for new, shorter drug regimens. This proposal addresses two critical impediments for TB drug development: (1) limited understanding of Mycobacterium tuberculosis (Mtb) phenotypes that persist during the late ?sterilizing? phase of TB treatment and are the cause of relapse and (2) lack of accurate markers of treatment response for use in clinical trials. Our central innovation is evaluating drug activity based on non-coding precursor ribosomal RNA (pre-rRNA), an unstable intermediate product that is transiently present during rRNA synthesis. This measures a fundamental bacterial process ? the rate of bacterial rRNA synthesis ? that is directly correlated with bacterial replication rate. Our experiments have suggested that Mtb subpopulations that persist after exposure to different antibiotics have a spectrum of rRNA synthesis and replication rates ranging from non-replicating (rifampin, bedaquiline) to continuing active replication (isoniazid, streptomycin, clofazamine). Our overall hypothesis is that the ratio of pre-rRNA to 23S rRNA will demonstrate that: (1) Mtb persister phenotypes range from non-replicating and dormant to actively replicating, depending on drug exposure and (2) the physiologic state of Mtb in human sputum is transformed by TB treatment. Our long-term objective is to develop novel surrogate markers for trials and provide critical biologic insights into persistence. Our scientific approach involves paired in vitro experiments and clinical studies. Using several complementary experimental approaches, we will first validate pre-rRNA to 23S ratios in vitro as accurate markers of Mtb replication state. We will then determine the impact of antibiotics (individually and in combination) on pre-rRNA to 23S ratios and Mtb replication. Finally, we will determine if pre-rRNA to 23S rRNA ratios independently predict Mtb growth in the sputum of Vietnamese and Ugandan patients during the intensive phase of TB treatment. This will lay a foundation for definitive testing in TB clinical trials. We believe this work has potential to lead to a new era in which TB drug trial decisions are based not on culture-based surrogates but rather on precise, in vivo molecular markers of the pathogen's physiologic state. This will accelerate design and evaluation of shorter and more effective TB drug regimens, and suggest new targets to be explored for future TB drug development.